DURON, AND ALOIS NOWOTNY. Relation of structure to function in bacterial 0 antigens. I. Isolation methods. J. Bacteriol. 85:418-426. 1963.-Six isolation methods, four previously described and two reported herein, were compared for the extraction of endotoxic 0 antigens from three different enterobacteria: Serratia marcescens Bizio, Salmonella typhosa 0901, and Escherichia coli K-12 lambda. Large variations were observed in the toxin yield, in the serologically reactive material yield, and in the nucleic acid-releasing abilities of the six methods wvhen applied to the different bacterial strains. Chemical analyses of the toxic 0 antigens included quantitative determination of the amino acids, long-and short-chain carboxylic acids, and carbohydrates. Comparisons of the nucleic acid content were also made. Although the
. Among the conserved residues in FucTs (including human, mouse, chicken, and zebra fish) are four cysteine (Cys) residues (Fig. 1). Two of these Cys residues are located near the N terminus and two near the C terminus of the catalytic domain. The Cys residues found at the C terminus also are conserved in FucTs from Caenorhabditis elegans (6). Among the human FucTs, FucTs III, V, and VI share substantial sequence homology. Within the catalytic domain only about 20 out of 300 amino acids vary among the three proteins. In addition, domain swapping experiments by our group (7) and Lowe and co-workers (9) have demonstrated that chimeric proteins composed of partial sequence from each of these FucTs are active, indicating that the minor differences in their amino acid sequences does not result in major alterations in their overall structure. Therefore, we have used two (FucT III and V) of these highly homologous proteins in the present study to evaluate the structure and functional significance of the highly conserved Cys residues.Protein chemistry experiments, coupled with mass spectrometry analyses, have been used to locate all peptides containing Cys residues in human FucT III, allowing them to be assigned either as being involved in a disulfide bond or as a free Cys residue, and identifying which Cys residues are bound to each other in disulfide linkages. The results support our (7) previously stated hypothesis that amino acids affecting acceptor substrate specificity of human FucT III and V, located near the N and C termini of the catalytic domain, are brought close together in space by disulfide bonds between these highly conserved Cys residues.To investigate the importance of these Cys residues, we have mutated each of these Cys residues in human FucT V and evaluated the activity and other properties of each resulting protein construct. The results demonstrate that these residues affect enzyme activity, but not the interaction of the protein with GDP-Fuc in three of the four cases. In the case of one of the mutant constructs (Cys 104 ) protein folding/stability is altered compared with the wild type protein.The amino acid sequences of FucTs have been predicted on the basis of cDNA sequences, but none of the amino acid se-
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